Department of Neuroscience, Vickie and Jack Farber Institute of Neuroscience, Thomas Jefferson University, Bluemle Life Sciences Building, Philadelphia, PA 19107, USA.
G3 (Bethesda). 2023 Nov 1;13(11). doi: 10.1093/g3journal/jkad221.
At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement, behaviors, and stimulus processing. The immense number and variety of neurons within the nervous system make discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via the expression of 2 independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Recent work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short-mStraw and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed the correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof of principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that the synaptic puncta number labeled by SynLight was comparable to the endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.
在突触处，化学性神经传递介导神经元之间的信息交换，从而产生复杂的运动、行为和刺激处理。神经系统内神经元的数量巨大且种类繁多，使得辨别单个神经元群体变得困难，这就需要开发先进的神经元标记技术。在果蝇中，Bruchpilot-Short 和 mCD8-GFP 分别标记突触前活性区和神经元膜，被广泛用于研究突触发育和组织。这种标记通常是通过单个双元表达系统表达 2 个独立的构建体来实现的，但当多个转基因由单个驱动子表达时，表达可能会减弱。最近的工作试图通过开发从单个转录本编码多个蛋白质的方法来规避这些缺点。自我切割肽，特别是 2A 肽，已成为完成此任务的有效序列。我们利用 2A 核糖体跳跃肽来设计一种构建体，该构建体可以从同一条 mRNA 产生 Bruchpilot-Short-mStraw 和 mCD8-GFP，我们将其命名为 SynLight。使用 SynLight，我们可视化了多个类别的嗅觉、视觉和运动神经元的假定突触活性区和膜，并观察到信号的正确分离，证实两种蛋白质是分别产生的。此外，我们通过量化嗅觉神经元中突触突点数和神经突体积来证明原理，并发现表达 SynLight 的神经元和分别表达两种转基因的神经元之间的突触密度没有差异。在神经肌肉接头处，我们确定 SynLight 标记的突触突点数与抗体染色标记的内源性突点数相当。总的来说，SynLight 是一种用于检查任何感兴趣的神经区域的突触密度的多功能工具，并允许提出关于突触发育和组织的新问题。
